Resources

Introduction to Zinc Sensing

In aqueous solutions of metal ions, the distinction is often made between "free" and "bound" metal ions.  This is an important concept in understanding the chemistry and biology of metal ions in solution. The term "free" is something of a misnomer, since essentially any ion will be solvated nearly all the time in aqueous solution, and likely to be at least weakly liganded by other ions and other molecular species that may also be present as well; others have proposed the use of alternative terms such as "mobile" or "labile"or "exchangeable" for such weakly bound metal ions.  

The important distinction is between weakly liganded metal ions and those more tightly bound, for instance bound as coenzymes to enzymes.   The free ion, bound by water, chloride, or other weak ligands, can rapidly exchange them for other, stronger ligands, and thus readily bind to other ligands and binding sites, which may bind more tightly and/or exchange more slowly(Stumm and Morgan 1996).  For example, a zinc ion in dilute aqueous solution will have water and anions such as chloride weakly bound to it, which adsorb and desorb rapidly (time frame of seconds, or faster).  When that zinc ion binds to apocarbonic anhydrase II in the active site (KD ≈ 4 pM), the dissociation rate constant is very slow (t1/2 ≈ months at room temperature without a catalyst), so the zinc-protein complex is very stable(Henkens and Sturtevant 1968).  Thus the free zinc is available to bind to other binding sites, while the protein-bound zinc is sequestered and essentially unavailable.  Thus an ordinary eukaryotic cell might have 100 μM total zinc ion, but less than one nM free zinc present in its cytoplasm, with the balance bound to proteins, amino acids, and other ligands present in the cell.  It is also important to note that there is a continuum of ligand affinities and kinetics in the cell, with both varying over a wide range(Bozym, Hurst et al. 2008).  Thus the zinc bound to (abundant) glutamate in the cell is not nearly as tightly bound at that to some proteins, and may exchange, albeit slowly.  

References:

Bozym, R., T. K. Hurst, et al. (2008). Determination of zinc using carbonic anhydrase-based fluorescence biosensors. Fluorescence Spectroscopy. L. Brand and M. Johnson. San Diego, Academic Press. 450: 287-309.

Henkens, R. W. and J. M. Sturtevant (1968). "The kinetics of the binding of Zn(II) by apocarbonic anhydrase." Journal of the American Chemical Society 90: 2669 - 2676.

Stumm, W. and J. J. Morgan (1996). Aquatic Chemistry: Chemical Equilibria and Rates in Natural Waters. New York, Wiley-Interscience.

Metal Ion Buffers

Introduction: Why use metal ion buffers?

Metal ion buffers are solutions of metal ions that are similar to, and share many advantages of, pH buffers.  Like pH buffers with protons, they “resist” changes in free metal ion concentration that would occur if, for instance, additional metal ion from another source somehow contaminated the sample under study.  Particularly when working with free metal ion concentrations below micromolar, the presence of contaminating metal ions in common buffer salts and water makes the use of metal ion buffers almost essential; please see our discussion of free vs. total metal ion concentration under Resources.  For instance, ACS Reagent Grade sodium chloride is specified as having up to 5 ppm by weight heavy metals as contaminants (determined as Pb). Thus making a physiological saline solution with 100 mM NaCl means it can contain roughly 150 nanomolar heavy metals, which are likely to interfere if one is trying to measure free zinc at the 100 picomolar level.  An analogous problem arises when buffer components or impurities bind the metal ion of interest: for instance, phosphate, citrate and Tris (tris(hydroxymethyl)aminomethane) all bind zinc with significant affinity. Thus adding one micromolar total zinc ion to a 20 millimolar pH 7.5 Tris buffer only leaves 1.3 picomolar free and bioavailable, a very large error.  

While the details of formulating metal ion buffers are beyond our scope here, the principle is actually straightforward: the metal ion buffer incorporates a fairly strong ligand together with a predetermined amount of the metal ion in question, such that a fixed proportion (preferably 99+%) of the metal is bound to the ligand. Thus addition or subtraction of metal ion causes a much smaller change in the free metal ion concentration than it would in the absence of the metal ion buffer.  For example, if we have a 20 mM pH 7.5 Bicine buffer with 0.62 picomolar free zinc ion present (1 uM total zinc ion added), addition of 100 nanomolar additional zinc ion only results in a free zinc ion concentration of  0.69 picomolar, a much smaller change than in the absence of the buffer.  

Use of Metal Ion Buffers to Calibrate Molecular Sensors

One of the best and most important uses of metal ion buffers is to calibrate fluorescence (and other) sensors.  The first reason this is important is that the response of the instrument one is using with the sensor varies a lot depending on the individual microscope, plate reader or fluorometer; i.e., even with a ratiometric sensor the actual numerical value of the ratio will vary from machine to machine.  The value of buffers for calibration will be evident if we consider the following: for a high affinity (KD ≈ 0.1 nM) fluorescence-based molecular zinc sensor, suppose we want to determine how big a fluorescence change corresponds to a particular value of free zinc concentration.  We might try to put 0.1 nanomolar (KD ) of ZnCl2 in a 4 ml cuvette (presuming there's no contaminating  zinc ion, other metals, or things that bind zinc, which is probably not true), but then we face another problem: if we then put (say) 1 nM of our sensor in the solution, it will largely scavenge up all the free zinc ( ≈ total zinc in this case) since the sensor concentration is 10x the free zinc concentration and also above KD.  In this case, the sensor fluorescence change will correspond to only 10% fractional occupancy (because that's all the zinc there is) rather than the 50% corresponding to KD: obviously a big error.  The problem of course, is that the free zinc concentration declines as it gets bound by the sensor: this is not so much the case in natural waters, and manifestly not the case in growth media, serum, or cell cytoplasm, which have abundant zinc-binding components which act as metal ion buffers. In addition, lowering the sensor concentration is seldom an option since the fluorescence signal drops correspondingly, and interfering fluorescence and electronic noise in the measurement grow in importance.  The metal ion buffer largely solves these problems for calibration because binding of the free zinc doesn't consume all the available zinc since 99+% is still bound by the buffer and the equilibrium merely shifts slightly as it is bound by the sensor. Moreover, enough sensor can be used to provide adequate signal to noise, with no concern about scavenging all available zinc. Pokegama MetalloBuffers are designed to make such calibrations easy.   

Pokegama Technologies MetalloBuffer products

Pokegama Technologies MetalloBuffersTM are formulated to provide reliable free zinc and copper concentrations for calibrating Pokegama and other metal ion sensors. We offer three different sets of metal ion buffers: 

1) Basic MetalloBuffers: These metal ion buffer sets are the simplest MetalloBuffer sets we offer.  They are designed for calibration of fluorescent or bioluminescent molecular zinc sensors (Pokegama’s and others on the market) in your microscope, fluorometer, or plate reader.  Since the lamp output, monochromator/filter throughput, and detector efficiency of any of these instruments varies with wavelength and from instrument to instrument, it is essential to calibrate the sensor in situ with known concentrations of free zinc.  This is especially true for non-ratiometric sensors such as TSQ, Zn-AF2, or  

2) Physiological Salts MetalloBuffers (in development): We have formulated MetalloBuffer sets in widely used physiological balanced salt solutions such as Earle’s Balanced Salts and Henke’s .  These buffers are intended for use with cells of all types to provide physiologically compatible media having known free zinc concentrations. 

3) MetalloBuffer Medium Supplements (in development):  Pokegama has formulated a series of zinc buffer supplements designed to be used together with common growth media such as DMEM to achieve preselected free zinc concentrations for use with mammalian cell cultures.  When used as directed, this permits the investigator to reliably carry out experiments with known, fixed free zinc concentrations to elucidate zinc effects, or calibrate sensors in situ.  Please note that although the supplements may be used with media that include fetal bovine serum, the variable amounts of zinc in serum make the derived values of free zinc in such media less accurate.  

Why use biosensors to measure free metal ions?

 Metal ion sensors: There are quite a variety of sensors of differing kinds designed to detect or quantify various metal ion species.  For the most part, we are interested in applications in cell biology, biochemistry, clinical analyses, bio/geochemistry, and environmental analyses, and thus primarily interested in measuring metal ion species dissolved in aqueous media.  Sensors capable of such analyses include devices which transduce the presence or level of the metal ion analyte initially as a change in electrical voltage or current, or an optical signal. Particularly for applications in biology, optical sensors are most useful because they are easily introduced into cells and compartments therein, and the analyte levels mapped by imaging in relation to the cell, tissue, or organism.  For the most part optical sensors for metal ions are molecules which exhibit changes in color, fluorescence, or chemi- or bioluminescence upon binding the analyte. Please see the discussion of “free” vs bound metal ions in Resources.  

Attributes/figures of merit for sensors: For sensors of all types figures of merit include  sensitivity, usually defined by the signal to noise ratio and detection limit; selectivity, meaning the ability to respond to the analyte of interest and not potential interferents; dynamic range, meaning the range of analyte levels which can be accurately quantified (and not the signal level difference between free and analyte-bound sensor);  and speed of response.  Other aspects may be important as well, including cell penetrability, targeting to organelles in cells, toxicity, susceptibility to washout or ejection from cells by multidrug resistance transporters (MDR) or other means, resistance to photobleaching, fluorescent “brightness”, and cost.   

Why use ratiometric sensors?

 There are a few reasons why investigators have largely switched to ratiometric fluorescence sensors (like those offered by Pokegama Technologies) from simple, intensity-based sensors.  Consider a classical, intensity-based sensor that exhibits an increase in fluorescence quantum yield when the analyte binds to it.  The measured fluorescence intensity F is a product of several factors: 

The issue is that although one might measure a change in fluorescence intensity F and attribute it to a change in the quantum yield of the sensor caused by binding of the analyte metal ion to the sensor, in fact the apparent intensity change might have been caused (wholly, or in part) from changes in almost any of the factors in the above expression.  So for instance, a decline in excitation intensity, or a decrease in thickness of different portions of the cell being measured, or washout of the sensor, or bleaching of the sensor/indicator, and/or quenching by an interferent, could all be misinterpreted as declines in the analyte concentration.   

Recognizing these issues, Walt, Tsien, Wolfbeis, and their colleagues developed so-called wavelength-ratiometric fluorescence sensors, where the analyte concentration was related to the relative strength of two emission (or excitation) bands, corresponding to analyte-free and -bound forms of the sensor; an example is shown below.